DE69426534T2 - TRANSFECTED CELL LINES THAT EXPRESS AUTOANTIGENS AND THEIR USE IN IMMUNOTESTS FOR DETECTING AUTOIMMUNE DISEASES - Google Patents

Abstract

This invention relates to improved in vitro immunoassay methods for detection of autoantibodies associated with autoimmune disease. More specifically, the invention relates to immunoassay methods which utilize a cell line stably transformed with a nucleic acid expressing an autoimmune antigen. This invention also relates to compositions comprising these cell lines and to kits containing such cell lines.

Description

Technical field of the invention

This invention relates to improved in vitro immunoassay methods for detecting autoantibodies associated with autoimmune diseases. More particularly, the invention relates to immunoassay methods that use a cell line stably transformed with a nucleic acid that expresses an autoimmune antigen. This invention also relates to compositions comprising these cell lines and kits containing such cell lines.

Background of the invention

Autoimmune diseases are those disorders of the immune system characterized by the production of antibodies that react with antigens in the patient's own tissues. Currently, more than 30 autoimmune diseases are recognized; these include many that have received much public attention, including rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus (SLE). The production of specific autoantibodies is associated with certain autoimmune diseases. For a description of the immune mechanisms and autoantibodies involved in autoimmune diseases, see Schwartz, R. S., et al. in Fundamental Immunology, second edition, Paul, W. E., editor, Raven Press, New York (1989), pp. 819-866.

Autoimmune diseases can be classified into the categories of organ-specific diseases and systemic diseases. Organ-specific autoimmune diseases affect a single organ, such as the thyroid, or a physiological system, such as the neuromuscular system. The autoantigens involved in organ-specific diseases are primarily antigens specific to an organ and may be associated with the pathology of the disease. For example, autoantibodies to thyroglobulin are observed in autoimmune thyroiditis and appear to be involved in the pathology of the disease. Systemic autoimmune diseases, on the other hand, affect multiple physiological systems. The autoantibodies involved in systemic autoimmune diseases are generally reactive with more ubiquitous autoantigens, which include a group of antigens found in the nucleus of cells. This latter group of autoantigens includes DNA, histones, and a number of ribonucleoproteins. See Schwartz, RS, et al., supra.

Detection and measurement of autoantibodies are used to diagnose and monitor a number of autoimmune diseases. For example, autoantibodies that are reactive with nuclear autoantigens are generally measured in the clinical laboratory using the antinuclear antibody (ANA) test. The ANA test is an indirect immunofluorescence test that uses a cell line or tissue section as a source of nuclear autoantigens. Nuclear fluorescence in the ANA test indicates the presence of nuclear autoantibodies. In addition, the fluorescence pattern observed can be related to the presence of autoantibodies that are reactive with specific nuclear antigens.

The ANA test is widely used to detect autoantibodies against nuclear antigens and is useful in the diagnosis of several systemic autoimmune diseases. However, there are problems associated with the ANA test that limit its diagnostic usefulness. In particular, it is difficult to find cell lines or tissue sources that produce sufficiently high levels of all of the desired nuclear autoantigens. Some nuclear autoantigens are produced only in small amounts in cell lines and tissue sources commonly used for ANA testing.

An example of such a nuclear autoantigen is Ro/SS-A. Autoantibodies to Ro/SS-A are associated with SLE, neonatal lupus erythematosus, Sjögren's syndrome, and other rheumatic diseases. Measurements of anti-Ro/SS-A autoantibodies are important in the diagnosis of these disorders. Therefore, cell lines that express higher levels of Ro/SS-A or other autoantigens that are normally present in low amounts are needed. For nuclear autoantigens such as Ro/SS-A, it would be particularly useful if a cell line that also expresses other nuclear autoantigens could be modified to overexpress Ro/SS-A so that the cell line can be used for an improved ANA test.

WO 91/17171, WO 89/09222, WO 92/20811 and Veldhoven et al (1992) Journal of Immunological Methods, Volume 151, pp. 177-189, discuss the use of recombinant autoantigens in assays for the detection of autoantibodies against autoantigens. Keech et al (1993) Journal of Autoimmunity, Volume 6, pp. 543-555 discusses the expression and maintenance of the human La(SS-B) core autoantigen in mouse cell lines.

The present invention provides a method for detecting anti-Ro/SS-A or anti-La/SS-B autoantibodies in a biological sample, comprising:

(a) contacting the biological sample with cells of a mammalian cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding a Ro/SS-A or La/SS- B autoantigen, wherein the autoantibodies are reactive with the Ro/SS-A or La/SS-B autoantigen and wherein the transfected cell line expresses the Ro/SS-A or La/SS-B autoantigen,

(b) incubating the biological sample with the cell line to form an autoantigen:autoantibody complex, and

(c) Detection of autoantigen: autoantibody complex.

The autoantigen:autoantibody complex is typically detected by immunofluorescence. The method can be used to detect rheumatic diseases. The present invention additionally provides a HEp-2 cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding a human Ro/SS-A autoantigen, wherein the transfected cell line overexpresses the autoantigen.

The present invention also provides a kit for detecting anti-Ro/SS-A or anti-LalSS-B autoantibodies in a biological sample, comprising:

(a) a human cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding a Ro/SS-A or La/SS-B autoantigen, and

(b) a labelled binding agent that specifically binds the Ro/SS-A or La/SS-B antigen, wherein the autoantibodies are reactive with the autoantigen and wherein the transfected cell line overexpresses the autoantigen.

The present invention also provides the use of a mammalian cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding a Ro/SS-A or La/SS-B autoantigen in a method for detecting anti-Ro/SS-A or anti-La/SS-B autoantibodies in a biological sample, wherein the autoantibodies are reactive with the Ro/SS-A or La/SS-B autoantigen and wherein the transfected cell line overexpresses the Ro/SS-A or La/SS-B autoantigen.

The autoantibodies may be associated with systemic autoimmune diseases such as neonatal lupus erythematosus. The antibodies may be associated with systemic lupus erythematosus or Sjögren's syndrome. Example antigens include the Ro/SS-A antigen. The Ro/SS-A antigen may be the 60 kd RoJSS-A or the 52 kd Ro/SS-A. Another example antigen is La/SS-B.

The cell line used may be a human cell line. The human cell line may be derived from HEp-2. The human cell line may be HEp/Ro 60 or HEp/Ro 52. The recombinant expression cassettes used in the invention may also comprise an early immediate promoter from human cytomegalovirus.

The cell line used in the invention typically overexpresses the autoantigens. The transfected cell line preferably overexpresses the autoantigens to improve the sensitivity of the assay.

The invention also provides kits for detecting autoantibodies in a biological sample comprising a cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding an autoantigen as described above. Exemplary cell lines are HEp/Ro 60 and HEp/Ro 52.

Definitions

"Antibody" refers to an immunoglobulin molecule that is capable of binding to a specific epitope on an antigen. Antibody may be a polyclonal mixture or monoclonal. Antibodies may be whole immunoglobulins derived from natural sources or from recombinant sources and may be immunoreactive regions of whole immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies may be in a variety of forms including, for example, Fv, Fab, and F(ab)Z, as well as single strands (e.g., Huston, et al., Proc. Nat. Acad. Sci. USA, 85: 5879-5883 (1988) and Bird et al., Science 242: 423-426 (1988), which are incorporated herein by reference). (Generally, see Hood, et al., Immunology, Benjamin, NY, 2nd ed. (1984) and Hunkapiller and Hood, Nature, 323: 15-16 (1986), which are incorporated herein by reference).

The term "epitope" refers to the binding site of an antibody. Epitopes are defined by sequences, but are also functionally defined by the ability of an antibody to bind and block the binding of a second antibody to the same site. This is routinely accomplished by competitive immunoassays and is called epitope mapping.

The term "autoantibody" as used herein refers to an antibody obtained from an individual or animal that is reactive with a normal cell antigen or normal cell antigens from the same individual or animal. The production of autoantibodies is generally associated with autoimmune diseases. Autoimmunity and the production of autoantibodies in human autoimmune diseases are discussed in detail in Schwartz, R. S., et al., supra.

The term "autoantigen" as used herein refers to an antigen present in normal individuals that binds to an autoantibody reactive. Examples of autoantigens include both organ-specific antigens such as thyroglobulin and ubiquitous cellular antigens such as DNA, histones, and ribonucleoprotein particles. A description of known autoantigens and their role in autoimmune diseases is described in Schwartz, RS, et al., supra.

The term "nuclear autoantigen" or "nuclear autoantigen" as used herein refers to an autoantigen present in the nucleus of the cell. Autoantibodies reactive with nuclear autoantigens are generally associated with systemic autoimmune diseases such as SLE and Sjögren's syndrome. Nuclear autoantigens include DNA, histones, and a number of ribonucleoproteins. See Schwartz, R. S., et al., supra.

The terms "Ro/SS-A antigen" or "Ro/SS-A autoantigen" refer to specific nuclear autoantigens. Ro/SS-A antigen is a 60 kd protein associated with small RNA molecules, Y1-Y5, found in both the nucleus and cytoplasm. There is also a related protein of 52 kd. Autoantibodies to the 52 kd Ro/SS-A protein are closely related to autoantibodies to the 60 kd Ro/SS-A protein. The above term refers to both the 60 kd protein and the 52 kd protein and to ribonucleoprotein complexes containing these proteins. The terms "60kd Ro/SS-A antigen,""60kd Ro/SS-A autoantigen," or "60kd Ro/SS-A protein" refer specifically to the 60kd protein or to ribonucleoprotein complexes containing this protein. The terms "52kd Ro/SS-A antigen,""52kd Ro/SS-A autoantigen," or "52kd Ro/SS-A protein" refer to the 52kd protein or to ribonucleoprotein complexes containing this protein. Autoantibodies to the Ro/SS-A antigen are associated with both SLE and Sjögren's syndrome. For a description of the structure of the Ro/SS-A antigen and its association with autoimmune diseases see Itoh, K., et al. (1991) J. Clin. Invest. 87: 177-186, Chan, EK, et al., (1991) J. Clin. Invest. 87: 68-76, and Harley, JB, et al. (1992) Rheumatic Disease Clinics of frL America 18 : 337-358, all of which are incorporated herein by reference.

The terms "La antigen," "La autoantigen," or "La/SS-B antigen" refer to a nuclear autoantigen containing an RNA polymerase III transcription termination factor that is expressed in the nucleus and is capable of ATP-dependent fusion of RNA/DNA hybrids. For a detailed description of the structure and function of the La antigen, see Gottlieb, E. et al. (1989) EMBO J. 8: 841-850 and Gottlieb, E., et al., EMBO J., 8: 851-861, both of which are incorporated herein by reference. The terms "La protein" or "La molecule" refer to the RNA polymerase III transcription termination factor described above. The La protein is associated with a variety of small RNAs, including the precursors of cellular SS RNA and tRNA, 75 RNA, and small cytoplasmic RNAs associated with the Ro/SS-A autoantigen. Autoantibodies to the La antiautogen are associated with both Sjögren's syndrome and SLE.

"Biological sample" as used herein refers to any sample obtained from a living organism or from a deceased organism. Examples of biological samples include body fluids and tissue samples.

"Nucleic acids" as used herein refers to either DNA or RNA. "Nucleic acid sequence" or "polynucleotide sequence" refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from 5' to 3' end. This includes self-replicating plasmids, transferable polymers of DNA or RNA, as well as non-functional DNA or RNA.

"Nucleic acid probes" may be DNA or RNA fragments. For example, DNA fragments are prepared by digesting plasmid DNA or by using PCR, or they are synthesized either by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett. 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103: 3185 (1981), both of which are incorporated herein by reference.

The term "overexpressed" or "overexpression" as used herein refers to an amount of protein expression that is greater than that which occurs naturally. For example, a transfected cell line is said to overexpress a particular protein if it expresses a greater amount of the protein than is expressed in the non-transfected parent cell line. This generally occurs when the cell line is transfected with a nucleic acid capable of expressing the particular protein molecule. For example, transfection of a HEp-2 cell line with a cDNA expressing the 60kd Ro/SS-A antigen as described in Examples 1-3 herein resulted in expression of the 60kd Ro/SS-A antigen at a level that was significantly greater than that found in the parent HEp-2 cells.

When referring to an autoantigen expressed by a transfected cell line, overexpression can be measured by determining the titer of a panel of antisera against both the non-transfected parent cell line and the transfected cell line in an indirect immunofluorescence assay as described in Examples 2 and 3 herein. Overexpression refers to an amount of autoantigen expressed in the transfected cell line that results in a titer at least 8-fold greater than that observed for the parent cell line when following the experimental protocol of Example 2. More preferably, an increase in titer of at least 16-fold is observed, even more preferably an increase in titer of at least 32-fold is obtained, even more preferably an increase in titer of at least 64-fold is observed, and most preferably an increase in titer of at least 128-fold or greater is obtained. See Table 1 for an illustration of the measurement of the increase in titer of a panel of autoantisera to determine overexpression of the Ro/SS-A autoantigen.

The term "recombinant expression cassette" refers to a recombinant DNA fragment operably linked to a promoter (which is either constitutive or inducible) suitable for ligation into an expression vector. The recombinant DNA fragment generally encodes a protein or a fragment of a protein. For example, a recombinant expression cassette may contain a DNA or cDNA molecule encoding a particular autoantigen protein.

The term "cell culture" refers to the enclosure of growing cells derived from a multicellular plant or animal, allowing the cells to remain viable outside of the original plant or animal. The term "cell line" refers to a line of cells cultured in cell culture. The term includes both immortalized cell lines and original (non-immortalized) cell lines.

The term "human cell line" refers to a cell line derived from human tissue and consisting of human cells. The term "Hep/Ro 60" refers to a HEp-2 cell line that has been stably transfected with a nucleic acid encoding the 60kd Ro/SS-A Antigen encoded. The term "Hep/Ro 52" refers to a HEp-2 cell line that has been stably transfected with a nucleic acid encoding the 52kd Ro/SS-A antigen.

Short description of the drawings

Fig. 1. The 60kd Ro/SS-A protein is more readily detected by indirect immunofluorescence in the nucleus of HEp-2 cells transfected with 60kd Ro/SS-A than in non-transfected cells. HEp Ro/60(A) and HEp-2(B) are stained with Mab 2G10. Hep/Ro 60(C) and HEp-2(D) are stained with anti-60kd Ro/SS A reference sera (CDC7) (original magnified 400x).

Fig. 2. Immunoblot showing that the transfected Ro/SS-A cDNA is expressed as a 60kd protein. Cell lysates were transferred to a nitrocellulose membrane and probed with a MAb specific for 60kd Ro/SS-A (2G10). Reactivity was detected using 125I protein A and autoradiography.

Fig. 3. Comparison of an IFA assay using HEp/Ro 60 with an ELISA assay. The immunofluorescence and ELISA assays were performed as described in Example 4.

Fig. 4. Immunofluorescence staining of HEp-2(A) and HEp/Ro 52(B) with anti-52kd Ro sera. Immunofluorescence was performed as described in Example 6.

Fig. 5. Immunoblot showing that the transfected Ro/SS-A is expressed as a 52kd protein. Cell lysates were run on 10% SDS-PAGE and transferred to nitrocellulose membranes and probed with autoantisera specific for 52kd Ro/SS-A. Reactivity was demonstrated using enhanced chemiluminescence and fluorescence imaging.

Fig. 6. Gene constructs. (A) Reconstruction of the human genome La gene. EcoRI fragments of 4, 4, 4.6 and 6.Bkb were isolated from two λ phage clones, λLa20.2, and subcloned into the plasmid pGEM-7Zf. During the construction of the gene, 40 base pairs of a polylinker sequence were introduced into an intron (P). The approximate locations of the exons are indicated as solid vertical bars. (B) Human La cDNA clone in the expression vector pEE6/HCMVIGS. Restriction endonuclease sites are indicated as follows: B, BamHI; E, EcoRI; K, Kpul, Xb, Xbal; Xh, Xhol.

Fig. 7. Nuclear localization of human La expressed in transfected mouse fibroblasts. Indirect immunofluorescence staining of mouse fibroblast line LTA-5 transfected with the human genomic La construct (top right panel) and untransfected LTA-5 (top left panel) with mAb A1 specific for human La; and staining of untransfected LTA-5 with normal human serum (bottom left panel) or a human autoantiserum containing anti-La and anti-60kd Ro activity (bottom right panel). showing endogenous mouse La expression. Magnification 400x.

Fig. 8. Expression of human La in transfected mouse fibroblasts. Western blot analysis in LTA-5 cells transfected with the human genomic (LTA-La g1-g3) and human cDNA (LTA-La cl) La genes. Lysates were transferred to a nitrocellulose membrane and probed with a patient autoantiserum (anti-La serum) that cross-reacts with mouse La and with a human La-specific mAb, A1 (anti-human La). Reactivity was developed using a peroxidase-coupled second antibody and enhanced chemiluminescence.

Description of the preferred embodiment

The present invention relates to improved in vitro immunoassay methods for detecting autoantibodies associated with autoimmune diseases. These immunoassay methods utilize cell lines transfected with nucleic acids expressing various autoantigens. Methods for transfecting cell lines with isolated nucleic acids encoding autoantigens are described herein. Methods are also described for detecting autoantibodies using a variety of immunoassay methods using the transfected cell lines.

A. Isolation of nucleic acids encoding autoantigens

To develop a transfected cell line to express a specific autoantigen, it is first necessary to obtain a gene encoding the autoantigen. DNA or cDNA molecule. The DNA encoding the autoantigen can then be incorporated into a suitable expression vector and transfected into a cell line to induce expression of the autoantigen by the cell line.

As described above, there are a variety of different autoantibodies that are associated with various autoimmune diseases. Such autoimmune diseases include Sjögren's syndrome, scleroderma, polymyositis, dermatomyositis, systemic lupus erythematosus, juvenile rheumatoid arthritis, ankylospondylitis, myasthenia gravis syndrome (antibodies against acetylcholine receptors), bullous pemphigoid (antibodies against basement membrane at dermis-epidermis junction), pemphigus (antibodies against mucopolysaccharide-protein complex or intracellular cement substance), glomerulonephritis (antibodies against glomerular basement membrane), Goodpasture syndrome, autoimmune hemolytic anemia (antibodies against erythrocytes), Hashimoto's thyroiditis (antibodies against thyroid), pernicious anemia (antibodies against intrinsic factor), idiopathic thrombocytopenic Purpura (antibodies to platelets), Graves' disease and Addison's disease (antibodies to thyroglobulin) and the like.

A variety of autoantibodies have been identified that are associated with various autoimmune diseases, including anti-thyroglobulin, anti-thyrotropin receptor, anti-thyroid microsomal peroxidase, anti-islet cell (64kd protein), anti-insulin, anti-insulin receptor, anti-mitochondrion (74kd protein), anti-acetylcholine receptor (α-subunit), anti-factor VIII; anti-IgG (Fc region), anti-C-1 inhibitor, anti-basement membrane, anti-intercellular cement substance, anti-myelin basic protein, anti-cytoskeletal proteins (e.g. vimentin, tubulin), anti-myelin glycoprotein, anti-I (erythrocyte membrane glycolipid), Anti-DNA, anti-ribonucleoprotein, anti-phospholipid (cardiolipin).

The binding specificities for autoantibodies include antigens present in nuclei, cytoplasm, cell membranes, plasma proteins, hormones, enzymes and receptors. Autoantigens include, for example, a wide variety of proteins, nucleic acids, phospholipids, lipoproteins, sugars and steroids. Ubiquitous autoantigens, present throughout the body, include DNA, histones, ribonucleoprotein particles, ribosome phosphoproteins, mitochondrial proteins, cardiolipins and various cytoskeletal proteins including actin, vimentin, tubulin and keratins. Organ-specific autoantigens include, for example, peptide hormones such as insulin, acetylcholine and thyrotropin receptors, pancreatic β-cells, thyroglobulin, red blood cell antigens, and platelet antigens. In some cases, the molecular determinant that an autoantibody recognizes is unknown. However, cDNAs encoding a number of autoantigens have now been cloned, including, for example, cytoplasmic ATPase/dATPase, La antigen, 60kd nuclear protein NH2-terminus, RoISS-A, 6Bkd nuclear polypeptide, U1 RNP, 7Skd nucleolus acidic protein, PM-Scl, nuclear topoisomerase I, Sc1-70, tRNA, alanyl tRNA synthetase, PL-12, epithelial cell cadherin, mitochondrial acetyltransferase, mitochondrial cytochrome 450dbl, β-cell glutamic acid decarboxylase, iodide peroxidase, and brush border gp 330. For a more detailed discussion of autoantibodies and autoantigens associated with autoimmune diseases, see Schwartz, R. S., et al., supra.

There are various methods for isolating DNA sequences encoding autoantigens. Techniques for nucleic acid manipulation of genes encoding these polypeptides, such as subcloning nucleic acid sequences encoding polypeptides into expression vectors, Probe labeling, DNA hybridization, transfection of cells, and the like, are generally described in Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, which is incorporated herein by reference. This manual is hereinafter referred to as "Sambrook, et al."

Those of skill in the art are knowledgeable about the various methods for isolating DNA and cDNA molecules encoding autoantigens. Briefly, nucleic acid sequences encoding an autoantigen can be isolated by probing a genomic or cDNA library. Genomic or cDNA libraries are generally prepared as described in Sambrook, et al.

There are a variety of methods for probing genomic or cDNA libraries. Nucleic acid probes are often used, particularly when DNA sequence information is available for nucleic acids encoding the autoantigen protein. For example, DNA is isolated from a genomic or cDNA library using labeled oligonucleotide probes specific for sequences in the DNA. Restriction endonuclease digestion of genomic DNA or cDNA containing DNA sequences encoding an autoantigen can be used to isolate the DNA encoding these proteins. The DNA sequences encoding a variety of autoantigens are known (see Schwartz, RS, et al., supra). In these cases, a panel of restriction endonucleases can be designed to result in cleavage of the DNA in the desired regions. After restriction endonuclease digestion, DNA encoding an autoantigen is identified by its ability to hybridize with nucleic acid probes, for example on Southern blots, and these DNA regions are Standard procedures familiar to those skilled in the art. See Sambrook, et. al.

The polymerase chain reaction can also be used to produce DNA encoding an autoantigen. Polymerase chain reaction (PCR) technology is used to amplify nucleic acid sequences of autoantigen polypeptides directly from mRNA, from cDNA, and from genomic or cDNA libraries. Appropriate primers and probes for amplifying DNA encoding an autoantigen are generated by analyzing the DNA sequences. Briefly, oligonucleotide primers are synthesized that are complementary to the two 3' boundaries of the DNA region to be amplified. The polymerase chain reaction is then carried out using the two primers. See PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., editors), Academic Press, San Diego (1990). Primers can be selected to amplify the entire region encoding a full-length autoantigen protein or to amplify smaller DNA segments, as desired.

Oligonucleotides for use as probes are chemically synthesized by the solid phase phosphoramidite triester method first described by Beaucage, S. L. and Carruthers, M. H., 1981, Tetrahedron Letts., 22(20): 1859-1862, using an automated synthesizer as described in Needham-VanDevanter, D. R., et al., 1984, Nucleic Acids Res., 12: 6159-6168. Purification of oligonucleotides is done either by native acrylamide gel electrophoresis or by anion exchange HPLC as described in Pearson, J. D. and Regnier, F. E., 1983, J. Chrom., 255: 137-149.

The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam, AM and Gilbert, 1980, in W., Grossman, L. and Moldave, D., editors, Academic Press, New York, Methods in Enzymology, 65: 499-560.

A variety of expression cloning methods using probes or assays for the expressed proteins can also be used to isolate nucleic acids encoding autoantigens. Expression cloning methods can be used with expression libraries. For example, a cDNA population can be ligated into vectors designed to allow transcription and translation of the coding region of the cDNA. The desired cDNA clone can then be identified by immunoscreening with an antibody or antiserum that recognizes the desired protein. A variety of immunoscreening methods using expression cloning are known to those skilled in the art. See Sambrook, et al. Immunological screening methods are particularly useful for isolating nucleic acids encoding autoantigens, because of the availability of autoimmune antisera or specific antibodies for use in screening.

Other methods known to those skilled in the art can also be used to isolate nucleic acids encoding autoantigens. See Sambrook, et al. for a description of other techniques for isolating DNA encoding specific protein molecules.

8. Transfection of isolated nucleic acids expressing autoantigens into cell lines

Expression of nucleic acids encoding autoantigens in cell culture will typically be achieved by operably linking the gene or cDNA to a promoter (which is either constitutive or inducible) and incorporating it into an expression vector.

The vectors are suitable for replication and integration of the desired cell lines. The particular promoter used in the construct is not essential to the invention; any strong promoter can be used to generate stable transfectants suitable for use in the invention. Exemplary promoters include promoters derived from human cytomegalovirus, metallothionine promoter, SV-40 early promoter, SV-40 late promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters proven effective for expression in eukaryotic cells. Typical expression vectors contain transcription and translation terminators, initiator sequences, and promoters useful for regulating expression of DNA encoding autoantigens. The vectors may also comprise generic expression cassettes containing at least one independent terminator sequence, sequences allowing replication of the plasmid in both eukaryotes and prokaryotes, i.e. shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems. The vectors preferably contain a marker to create a phenotypic trait for selection of transformed host cells, such as conferring resistance to antibiotics such as ampicillin or neomycin.

Isolated DNA or cDNA molecules encoding autoimmune antigens can be ligated to various expression vectors for use in transforming cell cultures. See Sambrook, et al., supra for a description of expression vectors and ligation procedures. The gene sequences for initiating transcription and translation of the DNA sequences encoding the autoantigens are selected to be compatible with the selected host cell. A variety of cloning vectors can be used, including those derived from viruses and plasmids. Examples of expression vectors include: suitable for use with mammalian cells are described in Examples 1, 4 and 8 herein.

Preferably, mammalian cell lines are used as host cells for transfection. A variety of different cell lines are desirable for transfection by different autoantigens. A list of readily available cell lines is described in ATCC Catalogue of Cell Lines and Hybridomas, 7th edition (1992), ATCC, Rockville, Maryland, USA, which is incorporated herein by reference. For example, human cell lines such as HEp-2, which are useful for use in the ANA assay, are desirable host cells for transfection with DNA encoding the core autoantigens. These transfected cell lines may be useful in developing improved ANA assays or assays that complement the ANA assay. For example, the HEp-Ro 60-transfected cell line described in Examples 1-3 herein may be useful as an improved cell line for the ANA assay because it expresses increased levels of the 60kd Ro/SS-A antigen. This cell line may be useful in an improved ANA assay that can detect Ro/SS-A autoantibodies with higher sensitivity. Alternatively, this cell line or other cell lines transfected with DNA expressing the 60kd Ro/SS-A antigen may be useful in developing a more specific assay for Ro/SS-A autoantibodies.

The host cells are competent for transformation or are made competent by various means. There are several well-known methods for introducing DNA into animal cells. These include: calcium phosphate precipitation, fusing the recipient cells with bacterial protoplasts containing the DNA, treating the recipient cells with liposomes containing the DNA, DEAE-dextran, electroporation, and microinjection of the DNA directly into the cells. See Sambrook, et al. for a detailed description of transformation methods.

The transformed cells are cultured by means well known in the art. Biochemical Methods in Cell Culture and Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc., (1977). A variety of immunoassay formats known to those skilled in the art can be used to select transfected cell lines that express or overexpress an autoantigen (see below). Cell lines that express or overexpress selected autoantigens due to transfection with a nucleic acid encoding the autoantigen can then be used to develop immunoassays.

C. Immunoassays for measuring autoantibodies

Autoantibodies reactive with a particular protein can be measured by a variety of immunoassay methods. For a review of immunological techniques and immunoassay techniques in general, see Basic and Clinical Immunology, 7th edition (D. Stites and A. Terr ed.) 1991. In addition, the immunoassays of the present invention using transfected cell lines expressing particular autoantigens can be performed in any of several configurations. These immunoassay configurations are discussed in detail in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida (1980), "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Laboratory Techniques in Biochemistry and molecular biology, Elsevier Science Publishers B. V. Amsterdam (1985), and Harlow and Lane, Antibodies, A Laboratory Manual, supra.

Immunoassays for measuring autoantibodies can be either competitive or non-competitive binding assays. In competitive binding assays, the sample analyte competes with a labeled analyte for specific binding sites on a capture agent that is attached to a solid surface. Preferably, the capture agent is a transfected cell line that overexpresses an autoantigen. In competitive binding assays, for example, the labeled analyte is a labeled antibody that competes for binding to the autoantigen. The concentration of labeled analyte bound to the transfected cell line is inversely proportional to the amount of free analyte present in the sample.

Non-competitive assays are typically sandwich assays in which the sample analyte is bound between two analyte-specific binding reagents. One of the binding agents is used as a capture agent and is bound to a solid surface. The second binding agent is labeled and is used to measure or detect the resulting complex by visual means or by instrumentation.

A number of combinations of capture agent and labeled binding agent can be used. Preferably, a transfected cell line overexpressing an autoantigen is used as the capture agent, and labeled anti-human antibodies specific for the constant region of human antibodies can be used as the labeled binding agent. Goat, sheep and other non-human antibodies specific for constant regions of human immunoglobulins (e.g., γ or u) are well known in the art.

Other proteins capable of specifically binding constant regions of human immunoglobulins, such as protein A or protein G, can also be used as the labeled binding agent. These proteins are normal components of the cell walls of streptococcal bacteria. They exhibit strong non-immunogenic reactivity with constant regions of immunoglobulins from a variety of species. See generally Kronval, et al., J. Immunol. 111: 1401-1406 (1973), and Akerstrom, et al., J. Immunol., 135: 2589-2542 (1985). The above immunoassays all use a solid phase separation technique in which one of the components of the immunoassay is attached to a solid support. In the above test formats, the solid support is preferably a transfected cell line attached to a suitable solid support such as a microscope slide or microtiter plate.

The immunoassay formats described above use labeled test components. The label can be in a variety of forms. The label can be coupled directly or indirectly to the desired component of the assay using methods well known in the art. The component can be labeled using any of several methods. Traditionally, a radioactive label containing 3H, 125I, 35S, 14C, 32P has been used. Non-radioactive labels include ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies that can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of association with the compound, stability requirements, and available instrumentation.

Non-radioactive labels can be attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently attached to the molecule. The ligand then binds to an anti-ligand molecule (e.g., streptavidin) that is either inherently detectable or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. A number of ligands and anti-ligands can be used. If a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any Hapten compound or antigenic compound can be used in combination with an antibody.

The molecules can also be directly conjugated to signal-generating compounds, for example by combining them with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinediones, e.g. luminol. For a review of various labeling systems or signal-generating systems that can be used, see U.S. Patent No. 4,391,904.

The labeled compounds can be detected by a variety of means. The means of detecting the labeled compound depends on the type of label used and the format of the immunoassay. For example, in an indirect immunofluorescence assay, a fluorescence microscope is used. In this type of assay, the transfected cell line is bound to a microscope slide and autoantibodies are bound to autoantigens expressed in the cell line. A fluorescently labeled binding agent capable of binding to the autoantibodies is then used to visualize the bound autoantibodies under the fluorescence microscope. See Examples 2 and 3 herein for an illustration of an immunoassay in an indirect immunofluorescence format. The term "immunofluorescence" as used herein refers to both direct and indirect immunofluorescence methods. Direct immunofluorescence methods are those in which the primary antibodies that are reactive with the antigen are labeled with a fluorophore.

The labeled compounds can also be detected by light microscopy, for example, when enzyme labeling is used in an immunoenzymatic method. In this type of assay, the transfected cell line can be attached to a microscope slide, and autoantibodies are bound to autoantigens expressed in the cell line. An enzyme-labeled binding agent is then bound to the autoantibodies bound to the immobilized cells. Subsequent incubation with a substrate and a color-producing compound results in the formation of a chromogen that can be visualized by light microscopy. For example, the binding agent can be a non-human antibody labeled with horseradish peroxidase that is capable of binding human immunoglobulins. Incubation of the horseradish peroxidase with, for example, hydrogen peroxide and 4-chloronaphthol results in the formation of an insoluble chromogen that can be visualized by light microscopy. While light microscopy is preferred, the chromogens generated by such immunoenzyme methods can also be determined by other optical methods in addition to light microscopy.

This invention also includes diagnostic kits for detecting the presence of autoantibodies, comprising a transfected cell line expressing or overexpressing an autoantigen, a container and instructions for performing the test.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, the preferred methods and materials will now be described. Unless otherwise indicated, the techniques used or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, procedures, and examples are illustrative only and not restrictive.

Examples Example 1: Transfection of HEp-2 with cDNA encoding the 60kd human Ro/SS-A antigen a) Gene cloning and constructs

The full-length cDNA encoding the 60 kd Ro/SS-A molecule was cloned from a human T cell line (Hutt-78) cDNA library by screening with a 300 base pair 5' cDNA probe obtained by polymerase chain reaction. The cDNA was sequenced and found to be identical to that described by Deutcher et al. (1988) Proc. Natl. Acad. Sci. USA 85: 9479-9483. For transfection, the cDNA was cloned into a mammalian expression vector under the control of the early immediate promoter from human cytomegalovirus (IICMV) (described in Thomsen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 659-663) and polyadenylation sequences. This expression vector is based on pRC/CMV (Invitrogen, San Diego, CA).

b) Transfections

HEp-2 cells (ATCC #LCL23) (Moore, AF, et al. (1955) Cancer Res. 15: 998) were maintained as monolayers in RPMI supplemented with 10% fetal calf serum, non-essential amino acids, glutamine, antibiotics, and 5 x 10⁻⁵M 2-mercaptoethanol (complete RPMI). Transfection was performed by the calcium phosphate precipitation method (see Margulies H., Evans GA, Ozato K., Camirini-Otero RD, Tanaka K., Apella E., Seidman JG (1983) J. Immunol. 130: 463-470). To facilitate the selection of drug-resistant transformants, the selectable marker gene pSV2 neo (see Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1: 327-341) was co-transfected with the vector containing the 60 kd Ro/SS-AcDNA. Stable transfectants, designated HEp-Ro 60, were selected in complete RPMI containing 6418 (Gibco, Grand Island, NY) at 0.4 mg/ml and subsequently cloned by limiting dilution prior to analysis for expression of human 60 kd Ro/SS-A.

Example 1: Expression and intracellular localization of the 60 kd Ro/SS-A antigen in transfected HEp-2 cells a) Immunofluorescence

Cell lines were grown overnight on multiwell slides, then fixed in a mixture of acetone and methanol (3:1) at -20ºC for 2 minutes, then air dried. 60kd Ro/SS-A protein was detected by incubating the monolayers with human sera diluted in phosphate buffered saline (PBS) or clean monoclonal antibody (MAb) culture supernatant for 30 minutes. After washing with PBS, the monolayers were incubated with fluorescein isothiocyanate-labeled sheep anti-human immunoglobulin or sheep anti-mouse immunoglobulin (Silenus, Australia) diluted 1:100 in PBS. The anti-60kd Ro/SS-A MAb, 2G10, was a gift from Dr. GJM Prujin (University of Nijmegen, the Netherlands). The anti-La/SS-B MAb, A1, was a gift from Dr. EM Tan and has been described previously (see Chan EKL, Tan, EM (1987), J. Exp. Med. 166 : 1627-1640). Endpoint titers were determined after two-fold serial dilution of sera at 1:100. Normal human sera were screened at a dilution of 1:100. Anti-Ro/SS-A positive sera used in this study were positive for anti-Ro/SS-A by countercurrent immunofluorescence (CIE) and negative for other specific properties by CIE and IF-ANA. Other sera included reference sera for Ro/SS-A, La/SS-B, nRNP, Sm, Scl-70 (CDC, Atlanta), PCNA, centromere, mitotic spindle, and sera from 20 healthy donors.

Indirect immunofluorescence (IF) staining of HEp-2 and 60kd Ro/SS-A-transfected HEp2 cells (HEp/Ro 60) showed the typical characteristic nuclear speckles in the HEp-2 cells and a dramatic overexpression of Ro/SS-A in the transfectants (Fig. 1). In the transfectants, expression was predominantly nuclear, with intense nucleolus staining. The cells with the highest staining intensity also showed weak cytoplasmic staining, detected with anti-Ro/SS-A-specific autoantisera. No surface staining of fixed, non-permeabilized cells with MAb or autoantisera with anti-Ro/SS-A specificity was observed (data not shown). The extent of overexpression of 60 kd Ro/SS-A varied across the cloned population, resulting in non-uniform staining intensity ranging from background levels of endogenous Ro/SS-A to intense staining, yielding titers up to 128-fold greater than the endogenous expression level. HEp-Ro 60 cells with the highest intensity staining represented approximately 10-15% of the population. This change in expression level is believed to be due to regulation of expression by the HCMV promoter, and has been observed in other transfection systems.

b) Immunoblotting

Whole cell extracts were prepared by lysing cells at 2 x 107/ml in sodium dodecyl sulfate (SDS) sample buffer containing dithiothreitol. After boiling for 3 min, proteins were separated by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels. Proteins were transferred to nitrocellulose (Amersham, UK) using a semi-dry transfer device (Novablot, Pharmacia LKB, Sweden). Nitrocellulose filters were blocked for 1 h in PBS containing 3% skim milk powder, then incubated for 1 h in sera diluted in wash buffer (PBS, 3% milk powder and 0.5% Tween 20). The nitrocellulose filters were washed five times in wash buffer, then probed with rabbit anti-mouse immunoglobulin (Dako, Sweden) to enhance the signal. Antibody binding was detected using 125I-protein A (Amersham, UK) and autoradiography. Pre-stained molecular weight markers (Bio-Rad, Richmond, CA) were used to determine molecular mass.

Western blot analysis of HEp/Ro 60 cells probed with anti-Ro/SS-A specific MAb, 2G10, showed an increase in the expression level of an immunologically identical 60kd protein (Fig. 2). The overexpressed Ro/SS-A polypeptide reacts with both monoclonal and polyclonal anti-Ro/SS-A antibodies and has a molecular weight identical to endogenous 60kd Ro/SS-A. The discontinuous nuclear speckle pattern on IF is in agreement with previous studies showing that the Ro/SS-A antigen is predominantly nuclear localized. Interestingly, prominent nucleolus staining was also observed, implying that the 60kd Ro/SS-A protein shuttles between the nucleus and the nucleolus. Variable cytoplasmic staining was also observed, which could represent true cytoplasmic localization or could be due to diffusion of the antigen into the cytoplasm during fixation.

Despite overexpression, no Ro/SS-A was detected on the surface membrane of HEp/Ro 60. This is consistent with the finding that mouse cells transfected with human La/SS-B do not express surface La/SS-B under conditions of overexpression, ultraviolet irradiation, and serum deprivation, as reported by others (see Examples 4 and 5).

Example 3: Immunofluorescence of human sera on HEp/Ro 60 cells and on the HEp-2 mother cells

Immunofluorescence using HEp/Ro 60 and the HEp-2 mother cells was performed as described for Example 2. The anti-Ro/SS-A positive sera used in this study were positive for anti-Ro/SS-A by countercurrent immunofluorescence (CIE), and negative for other specific properties by CIE and IF-ANA. Other sera included reference sera for Ro/SS-A, La/SS-B, nRNP, Sm, Scl-70 (CDC, Atlanta), PCNA, centromere, mitotic spindle, and sera from 20 healthy donors.

Transfected HEp-2 cells were analyzed for their ability to accurately, sensitively and specifically detect 60kd Ro/SS-A by IF. Twenty-four sera positive for anti-Ro/SS-A at CIE were titered against HEp-2 and HEp/Ro 60 cells at 2-fold dilutions starting at 1:100 (see Table 1). Twenty normal human sera showed no staining on HEp-2 and HEpfRo 60 cells at 1:100 dilution. The dramatic increase in the endpoint titer of anti-60kd Ro/SS-A sera was accompanied by a characteristic staining pattern, allowing the sensitive and specific detection of anti-60kd Ro/SS-A protein. The expression of other autoantigens detected by routine IF-ANA was analyzed. No distortion of cell morphology, change in localization or of the expression level was observed when HEp/60 or the mother cell. HEp-2 were stained by IF when reference sera to Sm, Scl-70, PCNA, centromere, mitotic spindle and mAb to La/SS-B (data not shown). Table 1: Indirect immunofluorescence of human anti-Ro/SS-A sera on mother HEp-2 cells and 60kd Ro/SS-A-transfected HEp-2 cells*

* Cultured cells were fixed in acetone and methanol and incubated on glass slides with two-fold dilutions of human serum.

+ Positive for anti-Ro/SS-A on countercurrent immunoelectrophoresis

HEp-2 cells transfected with a cDNA encoding the full-length 60kd Ro/SS-A.

§ CDC Anti-Ro/SS-A reference serum.

Example 4: Comparison of an IFA using a transfected cell line with an ELISA method for the detection of anti-Ro/SS-A autoantibodies

The ability of HEp/Ro60 to sensitively and specifically detect 60kd Ro-specific autoantibodies in an IFA assay was compared in an ELISA format with commercially available recombinant 60kd Ro protein. The preparation of the HEp-Ro 60 cell line is described in Example 1 herein.

Immunofluorescence was performed as described in Example 1 herein. Endpoint titers were obtained after two-fold serial dilution of sera at 1:100. Normal human sera were screened at a dilution of 1:100.

ELISA tests for the detection of anti-Ro antibodies. Microwell ELISA plates (Nung, Denmark) were coated with recombinant 60kd Ro (AMRAD, Australia) at IU/well diluted in 0.03 M sodium carbonate buffer, pH 9.6, and were incubated overnight at 4ºC. After blocking with 3% bovine serum albumin, the wells were incubated with serum duplicates diluted 1:500 for 1 hour at 37ºC, followed by washing with PBS/0.05% Tween 20. Bound IgG was detected using alkaline phosphatase conjugated Anti-human IgG (Sigma, St. Louis, USA) and Sigma 104 phosphatase substrate. Optical density values (OD 405 nm) greater than 3 SD above the mean of 60 normal controls were considered positive.

The anti-Ro positive sera used in this study were positive for anti-Ro autoantibodies by countercurrent immunoelectrophoresis (CIE) and negative for other specific properties by CIE and immunofluorescence. Sera from 20 healthy volunteers were used as normal control sera.

Twenty-four sera positive for anti-Ro by CIE were titered against HEp-2 and HEp/Ro60 cells at two-fold dilutions starting at 1:100. These sera were also screened by the recombinant 60kd ELISA at a dilution of 1:500 (see Figure 3). Twenty normal human sera showed no staining on the parent HEp-2 or HEp/Ro60 cells at a dilution of 1:100 (data not shown). A dramatic increase in the endpoint titer against HEp/Ro60 was demonstrated, indicating the increase in the sensitivity of these transfectants to parent cells to detect 60kd Ro. The recombinant 60kd Ro ELISA failed to detect 13 of the 24 sera with known reactivity for natural 60kd Ro protein. The failure to detect anti-60kd Ro antibodies by the recombinant 60kd Ro ELISA does not correlate with the low titer by immunofluorescence to HEp-2 or HEp/Ro60.

This example demonstrates the superior sensitivity of a human cell line transfected with human 60kd Ro (HEp/Ro60) compared to a recombinant 60kd Ro ELISA to detect autoantibodies against 60kd Ro. Sera for this study were assayed for their ability to detect natural Ro protein by countercurrent immunoelectrophoresis. selected. The transfected 60kd Ro is overexpressed in vivo in a eukaryotic expression system where production, post-translational modification and folding of the protein will produce a functional protein of native structure. In prokaryotic expression systems used to produce recombinant proteins, limited post-translational modifications occur and there is no guarantee that corrective native conformational folding will occur. The sensitivity of 60kd Ro to cleaving autoreactive epitopes suggests that many sera react with conformational determinants not present on recombinant proteins.

Example 5: Transfection of HEp-2 with cDNA encoding the human 52kd Ro/SS-A antigen a) Gene cloning and constructs

The full-length cDNA encoding the 52kd Ro molecule was cloned from a human T cell line cDNA library (Hutt-78) by screening with a full-length 52kd Ro cDNA probe obtained by polymerase chain reaction. For transfection, the cDNA was cloned into a mammalian expression vector analogous to pRc/CMV (Invitrogen). The plasmid contains the early immediate promoter from human cytomegalovirus (HCMV) (Thomsen, D. R., et al. (1984) Proc. Nat. Acad. Sci., USA 81: 659-663) and 3' polyadenylation sequences. The selectable marker was contained in a separate plasmid.

b) Transfections

HEp-2 cells were grown as monolayers in RPMI supplemented with 10% fetal calf serum, non-essential amino acids, glutamine, antibiotics and 5 x 10-5 M 2-mercaptoethanol (complete RPMI). Transfection was performed using the calcium phosphate precipitation method as described in Example 1 herein. To facilitate selection of drug-resistant transformants, the selectable marker gene pSV2 neo (Southern, PJ and Berg. P., supra) was co-transfected with the vector containing the 52kd Ro cDNA. Transfectants were selected in complete RPMI containing G418 (GibCo, Grand Island, NY) at 0.4 mg/ml and subsequently cloned by limiting dilution prior to analysis for expression of human 52kd Ro.

Example 6: Expression and intracellular localization of the 52kd Ro/SS-A antigen in transfected HEp-2 cells

Immunofluorescence was performed as described in Example 2. The 52kd protein was detected by incubating the monolayers with human sera diluted 1:100 in phosphate-buffered saline (PBS) for 30 minutes. The anti-Ro positive sera used in this study were positive for anti-Ro by countercurrent immunoelectrophoresis (CIE) and negative for other specific properties by CIE and IF-ANA. Immunoblotting was performed as described in Example 2.

Indirect immunofluorescence staining of HEp-2 and 52kd Ro-transfected HEp-2 (HEpIRo52) cells with autoantisera positive for the 52kd Ro protein revealed the typical nuclear speckles of the parent cell line and a dramatic overexpression of the 52kd Ro protein in the cytoplasm of HEp/Ro52 (Fig. 4). It is difficult to determine from indirect immunofluorescence whether there is localization in the nucleus of these transfectants as well as in the cytoplasm, but the pattern is predominantly cytoplasmic.

Western blot analysis of HEp/Ro52 probed with an anti-52kd Ro antiserum revealed a single overexpressed band of apparent molecular mass of 50kd that co-migrated with the endogenous 52kd Ro molecule (Fig. 5).

Example 7: Immunofluorescence of human sera on HEpIRo 52 cells: comparison with an ELISA test

Immunofluorescence was performed as described in Example 6 herein. The anti-Ro positive sera used in this study were positive for anti-Ro by countercurrent immunoelectrophoresis (CIE) and negative for other specific properties by CIE and IF-ANA.

Full-length 52kd Ro was expressed in Escherichia coli as a 6·histidine (6·His) fusion protein using the pQE vector and the QIA Express system (QIAGEN, CA, USA). Recombinant protein was prepared by metal chelate affinity chromatography in 8 M urea according to the manufacturer's instructions. Microwell ELISA plates (Nung, Denmark) were coated with 52kd Ro-6·His fusion protein or recombinant 60kd Ro (IU/well) (AMRAD, Australia) diluted in 0.03 M sodium carbonate buffer, pH 9.6, and incubated overnight at 4ºC. After blocking with 3% bovine serum albumin, wells were incubated with duplicate serum samples diluted 1:500 and washed with PBS/0.05% Tween 20, and bound IgG was detected using alkaline phosphatase-conjugated anti-human IgG (Sigma, St. Louis, USA) and Sigma 104 phosphatase substrate. Optical density (OD 405 nm) values that were more than 3 standard deviations (SD) above the mean of 60 normal controls were considered positive.

Twenty-three patient sera that were positive for Ro by countercurrent immunoelectrophoresis (CIE) were analyzed by IF on parent HEp-2 and HEpIRo52 cells for the detection of autoantibodies specific for the 52kd Ro protein. Sera were also analyzed by recombinant ELISA for reactivity against the 52kd and 60kd Ro proteins. Seventeen of the 23 sera were positive for 52kd Ro by ELISA, of which 13 showed cytoplasmic staining specific for the transfected 52kd Ro protein in HEp/Ro52 (Table 2). Sera that were negative for 52kd Ro by ELISA (60kd Ro monospecific sera and 10 normal control sera) were negative for 52kd Ro by IF on HEp/Ro52.

The ability to distinguish anti-52 and anti-60kd Ro activities should allow more precise classification and assignment of these autoantibodies to disease subgroups. These transfected cells will provide a simple diagnostic reagent for testing for the presence of autoantibodies to the 52kd Ro protein. Table 2. Comparison of immunofluorescence and ELISA for the detection of 52kd Ro

nd = not performed

(1) Patient sera positive for anti-Ro/SS-A on countercurrent immunoelectrophoresis

(2) Values 3 standard deviations above the mean of 20 normal were considered positive (OD > 0.250)

(3) Values 3 standard deviations above the mean of 20 normal were considered positive (OD > 0.182)

- No staining observed over endogenous expression

+ Weak cytoplasmic staining

+++ Strong cytoplasmic staining

Example 8: Transfection of mouse LTA-5 cells with cDNA and genomic DNA encoding the human La (SS-B) nuclear autoantigen a) Gene constructions

The human genome La-encoding plasmid pLa15.8 (Fig. 6A) was constructed in four steps from two Charon 3A λ phage clones, λLa2.1 and λLa26.2 (see Chambers, J. C., et al. J. Biol. Chem. 263: 18045-18051), which were a gift from Dr. J. Keene. The EcoRI fragments, 4.6 kb and 6.8 kb, from the phage were subcloned into the cloning vector pGEM-7Z (Promega, Madison, WI, USA). The 3' EcoRI site of the 4.4 kb subclone was removed by partial EcoRI digestion and Klenow deletion repair of the DNA ends. The 4.6 kb EcoRI fragment was then cloned with the correct orientation into the 5' EcoRI site of the 4.4 kb subclone. This larger fragment (9.0 kb) was removed by initial digestion of the XbaI polylinker restriction site followed by Klenow repair and ClaI digestion. This 9.0 kb fragment was then directionally cloned into the BamIII site after Klenow repair and ClaI digested polylinker site of the 6.8 kb subclone. The final genome clone (pLa15.8) contained in the vector pGEM7-Zf contains 40 base pairs of polylinker sequence inserted between the 4.4 kb and 6.8 kb EcoRI fragments.

Plasmid pCTLa (Fig. 6B) encodes a human La cDNA under the control of the human cytomegalovirus early immediate promoter. For this construction, a human La cDNA lacking 90 base pairs of 5' non-coding sequence was first cloned from a polymerase chain reaction product of a full-length La cDNA clone (see McNeilage, L. J., et al (1990), J. Immunol. 145: 3829-3835). A KpnI/EcoRI fragment containing 1097 nucleotides of the La cDNA was then replaced with the corresponding fragment from the original cDNA clone. The remainder of the reconstructed cDNA clone was sequenced to ensure that there were no polymerase chain reaction-derived substitutions. The resulting BamHI/EcoRI fragment containing the full-length La cDNA after Klenow repair was subcloned into a XbaI-Klenow-repaired cloning site of the expression vector pEE6/HCMV/GS (Celltech, UK).

b) Transfections

LTA-5 cells were maintained as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, nonessential amino acids, glutamine, antibiotics, and 5 x 10-5 M 2-mercaptoethanol (complete DMEM). Transfection was performed as described in Example 1 herein. To facilitate the selection of drug-resistant transformants, the selectable marker gene pSV2nco (see Southern, P. J., and Berg, P. (1982) J. Mol. Appl. Genet. 1: 327-341) was cotransfected with either the reconstructed human La genome gene (pLa15.8) or the cDNA construct (pCTLa). Transfectants were selected in complete DMEM containing G418 (Gibco, Grand Island, NY, USA) at 0.2 mg/ml and subsequently cloned by limiting dilution prior to analysis for human La expression.

Example 9: Localization and expression of human La antigen in transfected mouse fibroblasts a) Immunofluorescence

Cell lines were grown overnight on multiwell slides, then fixed in a mixture of acetone and methanol (3:1) for 2 minutes at -20ºC and then air dried. La protein was detected by incubating the monolayers with human sera or ascites fluid containing monoclonal antibody (mAb) diluted 1:100 in phosphate buffered saline (PRS) or clean supernatant from mAb culture for 30 minutes. After a PBS wash, the monolayers were incubated with a 1:100 dilution of fluorescein isothiocyanate-labeled [F(ab)2] sheep anti-human immunoglobulin or [F(ab)2] sheep anti-mouse immunoglobulin (Silenus, Australia). Monoclonal antibodies A1, A2, and A3 were a gift from Dr. E. M. Tan, and SW1, SW3, and SW5 were a gift from Dr. D. Williams and were described previously (see Chan, E. K. L., et al. (1987) J. Exp. Med. 166: 1627-. 1640 and Smith, P. R., et al. (1985) J. Immunol. Med. 77: 63-76). Human serum with anti-La activity came from a patient with primary Sjögren's syndrome and also contained activity against the 60 kDa Ro antigen. Serum with anti-60 kDa Ro activity but no anti-La specificity came from a patient with systemic lupus erythematosus.

Selectable marker genes were co-transfected with either the cDNA or the genomic human La constructs to generate stable, drug-resistant transfectants that independently express these forms of human La. After culture in selection media, transfected mouse fibroblasts were cloned by limiting dilution and analyzed by indirect immunofluorescence to determine the localization of the human La protein. Permeabilized transfectants, stained with specific anti-human La monoclonal antibodies, including mAb A1, revealed predominantly coarse nuclear speckles and fine nuclear granules. Weak, diffuse cytoplasmic staining was also observed. This pattern of La distribution was indistinguishable from that of endogenous mouse La stained with patient autoantibodies cross-reactive with mouse La antigen. The human-derived LacDNA transfectants and human-derived La genomic DNA transfectants showed an identical immunofluorescence pattern (see Fig. 7).

There was no detectable expression of human La protein on the cell surface of untransfected LTA-5 cells or any of the human La transfectants when assayed by indirect immunofluorescence of live cells by microscopy or by flow cytometry using anti-human La-specific mAbs or patient anti-La autoantisera. Surface expression of human La or endogenous mouse La could not be demonstrated following treatment of these human La transfectants under conditions reported to induce surface expression of endogenous La antigen in other cell types. The transfectants were exposed to a wide range of UVB irradiation doses (0, 1, 10, 100, and 500 m3/em2) and analyzed for expression of surface La of human origin by flow cytometry after 1, 4, or 20 hours of culture. Culture for 48 hours under conditions of serum starvation (0.5% FCS), followed by release from synchrony by addition of 10% concanavalin A-stimulated T cell supernatant (Baboonian, C., et al., (1989) Clin. Exp. Immunol. 78: 454-459) did not induce surface expression of human or mouse La as determined by flow cytometry. Mouse fibroblasts may not have all the necessary requirements for surface La expression; however, these data suggest that more tissue-specific or disease-specific factors other than relative overexpression of La is responsible for the surface expression observed by others.

b) Immunoblotting

Immunoblotting was performed as described in Example 2 herein. More specifically, Western blot analysis of La expression in LTA-5 cells transfected with human genomic (LTA La g1-g3) and human cDNA (LTA-La c1) La genes was performed. Lysates were transferred to a nitrocellulose membrane and probed with a patient autoantiserum (anti-La serum) that cross-reacts with mouse La and with a human-derived La-specific mAb, A1 (anti-human La). Reactivity was developed using a peroxidase-coupled second antibody and enhanced chemiluminescence fluorography.

Western blot analysis showed that human La in transfected mouse cells had the correct apparent molecular mass, since it co-migrated with La expressed in human cells and was clearly distinguishable by electrophoretic migration from the endogenous mouse La antigen, which runs at a lower apparent molecular mass. The human La antigen expressed in transfected mouse fibroblasts reacted with a human autoantiserum and with anti-La mAbs specific for human La protein. Human La protein expressed from genomic and cDNA constructs was similar in apparent molecular mass in most clone isolates (e.g., clones g3 and g1). However, in some transfectant clones expressing the genomic human La gene product (e.g., clones g1 and g2), bands for higher molecular weight protein were detected by Western blot analysis. The higher molecular weight forms of human La were detected by several human La-specific mAbs, SW 1 and SW3, and to a lesser extent by the patient anti-La antisera (see Fig. 8).

It is to be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes will occur to those skilled in the art in light thereof.

Claims (30)

1. A method for detecting anti-Ro/SS-A or anti-La/SS-B autoantibodies in a biological sample, comprising (a) contacting the biological sample with cells of a mammalian cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding a Ro/SS-A or La/SS-B autoantigen, the autoantibodies being reactive with the Ro/SS-A or La/SS-B autoantigen and the transfected cell line expressing the Ro/SS-A or La/SS-B autoantigen, (b) incubating the biological sample with the cell line to form an autoantigen:autoantibody complex, and (c) Detection of the autoantigen:autoantibody complex. 2. The method of claim 1, wherein the autoantibodies are associated with systemic lupus erythematosus or Sjögren's syndrome. 3. The method of claim 2, wherein the autoantigen is a Ro/SS-A antigen. 4. The method of claim 3, wherein the Ro/SS-A antigen is Ro/SS-A of 60kd. 5. The method of claim 3, wherein the Ro/SS-A antigen is Ro/SS-A of 52kd. 6. The method of claim 2, wherein the autoantigen is a La/SS-B antigen. 7. The method of claim 1, wherein the cell line is a human cell line. 8. The method of claim 7, wherein the human cell line is derived from HEp-2. 9. The method of claim 7, wherein the human cell line is HEp/Ro 60. 10. The method of claim 7, wherein the human cell line is HEp/Ro 52. 11. Method according to one of claims 1 to 10, in which the autoantigen-autoantibody complex from step (c) is detected by means of immunofluorescence. 12. The method of any one of claims 1 to 11, wherein the recombinant expression cassette further comprises a human cytomegalovirus early immediate promoter. 13. The method according to any one of claims 1 to 12, wherein the transfected cell line overexpresses the autoantigen. 14. HEp-2 cell line stably transfected with a recombinant expression cassette comprising a nucleic acid, encoding a human Ro/SS-A autoantigen, wherein the transfected cell line overexpresses the autoantigen. 15. The cell line of claim 14, wherein the cell line is HEp-Ro 60. 16. The cell line of claim 14, wherein the cell line is HEp-Ro52. 17. A cell line according to any one of claims 14 to 16, wherein the recombinant expression cassette further comprises a human cytomegalovirus early immediate promoter. 18. 1< it for the detection of anti-Ro/SS-A or anti-La/SS-B autoantibodies in a biological sample, comprising (a) a human cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding a Ro/SS-A or La/SS-B autoantigen, and (b) a labelled binding agent that specifically binds the Ro/SS-A or La/SS-B antigen, wherein the autoantibodies are reactive with the autoantigen and wherein the transfected cell line overexpresses the autoantigen. 19. The kit of claim 18, wherein the Ro/SS-A antigen is Ro/SS-A of 60kd. 20. The kit of claim 18, wherein the Ro/SS-A antigen is Ro/SS-A of 52kd. 21. Kit according to one of claims 18 to 20, wherein the human cell line is derived from HEp-2. 22. The kit of claim 21, wherein the human cell line is HEp/Ro 60. 23. The kit of claim 21, wherein the human cell line is HEp/Ro 52. 24. Use of a mammalian cell line stably transfected with a recombinant expression cassette comprising a nucleic acid encoding a Ro/SS-A or La/SS-B autoantigen in a method for detecting anti-Ro/SS-A or anti-La/SS-B autoantibodies in a biological sample, wherein the autoantibodies are reactive with the Ro/SS-A or La/SS-B autoantigen and wherein the transfected cell line expresses the Ro/SS-A or La/SS-B autoantigen. 25. Use according to claim 24, wherein the autoantibodies are associated with systemic lupus erythematosus or Sjögren's syndrome. 26. Use according to claim 24, wherein the Ro/SS-A autoantigen is Ro/SS-A of 60kd or Ro/SS-A of 52kd. 27. Use according to claim 24, wherein the mammalian cell line is a human cell line. 28. Use according to claim 27, wherein the human cell line is derived from HEp-2. 29. Use according to claim 28, wherein the human cell line is HEp/Ro 60 or HEp/Ro 52. 30. Use according to claim 24, wherein the recombinant expression cassette further comprises a human cytomegalovirus early immediate promoter.